Labelled ascorbic acid derivatives

ABSTRACT

Compounds of formula (I) where: X&lt;1 &gt;is OH or SH or NH2 or -L-Z; X&lt;2 &gt;and X&lt;3 &gt;are the same or different and each is H, C1-4 alkyl, benzyl a protecting group or -L-Z, X&lt;4 &gt;is H or C1-4 alkyl, L is a linker comprising a chain of 0-10 atoms, Z is a group comprising a detectable moiety which comprise at least one detectable moiety are useful in the diagnosis and prognosis and radiotherapy of metastatic bone disease.

FIELD OF INVENTION AND BACKGROUND TO THE INVENTION

The present invention relates to a class of compounds useful in thediagnosis or radiotherapy of metastatic bone disease, pharmaceuticalformulations containing them, their use in the diagnosis of disease andmonitoring of disease progression and treatment, and methods for theirpreparation.

CURRENT BONE IMAGING AGENTS

Bone is a common site of metastatic disease with around 70-80% of breastand prostate cancers metastasising to bone. Lung, thyroid, kidney andbladder cancers can also metastasise to bone. Once tumour cells areimplanted in the bone marrow they release biochemical mediators whichactivate osteoblasts. The osteoblastic response detected on a bone scanis a secondary response. Osteoblasts are bone-producing cells implicatedin the pathology associated with metastatic bone disease. Activatedosteoblasts produce large quantities of collagen that, in addition toits structural role, is important in osteoblast differentiation.

Diagnosis of metastatic bone disease may be achieved by one of fourmethods—radiography, CT scanning, radioisotope bone scan or MRI. Theradioisotope bone scan has been the standard initial imaging method forthe past 25 years. The usual tracer for bone scans is ^(99m)Tc-methylenediphosphonate (^(99m)Tc-MDP). ^(99m)Tc-HMDP(hydroxy-methylenediphosphonate) and ^(99m)Tc-HEDP(1-hydroxyethyl-1,1-diphosphonate) may also be used. These agents havebroadly similar characteristics. Around 550-750 MBq (15-20 mCi) isinjected and high bone uptake (30-50% of the injected dose) occurswithin 2 hours. Scans are typically carried out 3-4 hr postadministration of agent, due to slow clearance from the blood and/ortissue. Whole body imaging (anterior/posterior) with an acquisition timeof 20-30 min produces images of high quality, good resolution and highsensitivity/specificity.

^(99m)Tc-MDP is adsorbed onto the calcium of hydroxyapatite in bone.This process is influenced by the levels of osteoblastic activity and byskeletal vascularity. There is preferential uptake at sites of activebone formation, and the amount of accumulation is sensitive to the levelof blood flow. The bone scan therefore reflects the metabolic reactionof bone to the disease process, regardless of whether the metabolicactivity is neoplastic, traumatic or inflammatory in nature. Thus, thetracer accumulates at any site of elevated bone turnover and the scan istherefore very non-specific.

Osteoblastic metastases resulting in hot spots are detected regardlessof size but a cold (photopenic) spot, caused as a result of osteolyticdisease, has to reach a certain size to be detected.

The general advantages of the radioisotope scan are a large field ofview, low cost, low morbidity, high sensitivity for detection ofskeletal metastases, ease of performance on any patient and relativelylow total body dose.

DISADVANTAGES AND PROBLEMS ASSOCIATED WITH CURRENT RADIOISOTOPE BONEIMAGING AGENTS

Tracer accumulation may occur at any skeletal site with an elevated rateof turnover and in this case does not provide functional or vascularinformation. As the bone scan has low specificity, the nature of anabnormality cannot be determined from the scan, hence benign andmalignant lesions often cannot be distinguished. The technique is alsoanatomically imprecise. Binding to bone can still occur after tumourcells are dead as collagen is still produced. Consequently there is nodistinction between bone healing and tumour progression, with the resultthat it is difficult to monitor effects of treatment. An increase in theuptake of ^(99m)Tc-MDP due to bone healing can be seen up to 6 monthsafter treatment and is known as the flare response.

There is no net production of collagen in osteolytic disease, hencefalse negatives occur—some or all lesions are missed. Such negativescans need to be re-evaluated with clinical and lab findings. If theseare non-conclusive then radiography is used, if this is stillnon-conclusive then bone biopsy or MRI are used.

The low specificity of ^(99m)Tc-MDP means the nature of the abnormalitye.g. benign vs malignant lesion cannot be detected. In a patient withknown primary tumours, multiple hot spots in the bone scan indicatemetastases. However 50% of these hot spots could be other non-metastaticlesions. Therefore a lack of specificity observed with ^(99m)Tc-MDPmeans that positive scans often have to be accompanied by radiographiccorrelation (a positive radiograph confirms the presence of metastasesas the bone scan is more sensitive, but a negative radiograph does notrule them out).

MRI is sometimes chosen, mainly due to its ability to demonstrateabnormalities in bone marrow. However, MRI often cannot distinguishbetween changes that are due to treatment, fracture and tumour and isless well suited to scanning long bones.

Despite the problems associated with the current radioisotope boneimaging agents, their unique features make them the first choice forscreening for metastases in a symptomatic patients. However, a negativescan should always be re-evaluated with clinical and laboratory findingsdue to the possibility of false negatives. Furthermore, the possibilityof a non-metastatic cause of an abnormal scan always needs to beconsidered. Non-conclusive findings generally lead to supplementaryexamination with radiography. If diagnosis is still unclear, bone biopsyor MRI will be performed.

There is therefore a need for a diagnostic imaging agent which hasspecificity for metastatic bone lesions (as opposed to other lesiontypes), and which can give clinically useful information in a singleimaging protocol, without the need for additional testing.

Skeletal metastases may respond to chemotherapy or hormone therapy usedto treat the primary tumour. They may also respond to radiation or toagents designed to block bone resorption such as the new class ofbisphosphonate (BP) drugs. Bisphosphonates have potent inhibitoryeffects on bone resorption and are the treatment of choice forhypercalcaemia of malignancy. Treatment can lead to a reduction in thenumber and rate of skeletal complications in multiple myeloma andadvanced breast cancer and can delay the onset of progressive disease inbone following palliative chemotherapy in breast cancer and myeloma. BPsalso relieve metastatic bone pain in around 50% of patients but thisrequires intravenous injection as BPs are not potent enough and nottolerated well when taken orally. Response to treatment can be measuredby biochemical markers e.g. excretion of collagen cross-links.Radioisotopes are also used in the treatment of bone metastases[Ben-Josef & Porter, Ann Med. 29, 31-35, (1997); Lewington, Phys MedBiol. 41, 2027-2042 (1996)]. ⁸⁹Sr has been successfully used in painpalliation. Other bone-seeking isotopes include ³²P (side effect ofmyelotoxicity), ¹⁵³Sm (complexed with EDTMP) and ¹⁸⁶Re (complexed withHEDP).

¹⁴C and ³H-labelled ascorbic acid derivatives are known. Yamamoto et al[Appl. Radiat. Isot. 43, 633-639 (1992)] have described the preparationof 6-deoxy-6-[¹⁸F]fluoro-L-ascorbic acid (¹⁸F-DFA), i.e. an ascorbicacid derivative labelled with the positron emitting isotope [¹⁸F] vianucleophilic displacement of a cyclic sulfate with fluoride ion. Thebiodistribution of this compound has been studied in rats andfibrosarcoma-bearing mice. Yamamoto et al [Radioisotopes, 44, 93-98(1995)] have also studied the biodistribution of ¹⁸F-DFA in Wistarnormal rats, ODS rats unable to synthesise ascorbic acid, and Wistarmale rats implanted with RG-G6 glioma intracerebrally, and [Nucl. Med.Biol., 23, 479-486 (1996)] the in vivo uptake and distribution of¹⁸F-DFA in rat brains following postischemic reperfusion.

The bone uptake reported for ¹⁸F-DFA is very low and there is nosuggestion that labelled ascorbic acid derivatives could be useful foreither bone imaging in general, or metastatic bone disease imaging inparticular. In addition, ¹⁸F has a half life of 1.8 hours, and istherefore only usable for a few hours (including synthesis andpurification time). Hence any clinical use of such PET (positronemission tomography) agents is limited to a very restricted number ofmedical sites which possess a cyclotron on site.

SUMMARY OF THE INVENTION

The invention includes diagnostic agents for the detection andmonitoring of metastatic bone disease as well as radiotherapy of suchdisease. The agents comprise a modified ascorbic acid labelled with adetectable moiety suitable for external imaging (e.g. by scintigraphy orMRI), such as a radionuclide or a paramagnetic metal ion.

Unmodified ascorbic acid has the formula:

The agents of the present invention act by accumulating in osteoblastcells present at sites of increased bone turnover. These sites includeareas of hyperproliferation associated with metastatic bone disease, aswell as other bone pathologies. As the ascorbic acid derivatives areonly taken up by osteoblasts at active lesions, they are of highdiagnostic and prognostic value for osteoblastic lesions and allow rapidmonitoring of disease progress. The use of ascorbates may also preventthe occurrence of false negative scans through visualisation of lyticlesions due to associated osteoblastic activity and also allow earlydiagnosis of small lesions due to the specific uptake mechanism. Uptakeinto normal bone will occur and will be useful for localising the lesionsite, where uptake will be greatly increased.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a compound of formula:

where: X¹ is OH or SH or NH₂ or -L-Z;

X² and X³ are the same or different and each is H, C₁₋₄ alkyl, C₁₋₄fluoroalkyl, benzyl, a protecting group or -L-Z;

X⁴ is H or C₁₋₄ alkyl;

L is a linker comprising a chain of 0-10 atoms;

Z is a group comprising a detectable moiety;

provided that the compound comprises at least one detectable moiety.

X¹ is preferably -L-Z. X² and X³ are preferably H or C₁ alkyl, mostpreferably both X² and X³ are H. X⁴ is preferably H or C₁ alkyl, mostpreferably H. The linker L is suitably a chain of 0-10 atoms of formula(A)_(m)

where: A is —CR₂—, —CR═CR—, —C≡C—, —NRCO—, —CONR—, —O(CO)—, —(CO)O—,—SO₂NR—, —NRSO₂—, —OCR₂—, —SCR₂—, —NRCR₂—, a C₄₋₈ cycloheteroalkylenegroup, a C₄₋₈ cycloalkylene group, a C₅₋₁₂ arylene group or a C₃₋₁₂heteroarylene group;

m is an integer of value 0 to 10;

each R group is independently chosen from H, C₁₋₄ alkyl, C₁₋₄ alkenyl,C₁₋₄ alkynyl, C₁₋₄ alkoxyalkyl or C₁₋₄ hydroxyalkyl.

L preferably comprises a 0-4 atom chain.

Preferred compounds have the defined stereochemistry shown:

with X¹—X⁴, L and Z as defined above.

The “detectable moiety” is a substance suitable for external imagingafter human administration and can be a radionuclide, where theradionuclide is a gamma-emitter that emits gamma radiation that canpenetrate soft tissue, a beta-emitter or a low energy X-ray emitter.Radionuclides which are positron emitters such as ¹¹C and ¹⁸F areoutside the scope of the present invention. ³H and ¹⁴C are not suitableradioisotopes for either external imaging or radiotherapy, and are hencealso outside the scope of the present invention. The detectable moietycan also be: one or more hyperpolarised atom(s) such as the ¹³C carbonatom of a ¹³C-enriched compound for MRI imaging; a paramagnetic moietyas a contrast agent for MRI (e.g. certain metal ions such asgadolinium(III), or manganese (II)); a radiopaque moiety such asiopamidol for X-ray contrast imaging (computer assisted tomography) oran ultrasound contrast agent. Preferably, the detectable moiety iseither a radionuclide γ-emitter such as ¹²³I, ^(99m)TC, ¹¹¹In, ^(113m)Inor ⁶⁷Ga or a hyperpolarised material. Most preferred radionuclideγ-emitters are ¹²³I and ^(99m)Tc, especially ^(99m)Tc. It is alsoenvisaged that certain radionuclides will confer useful radiotherapeuticproperties on the labelled ascorbic acid. Thus for example ⁹⁰Y, ⁸⁹Sr,¹⁸⁶Re, ¹⁸⁸Re, ¹²⁵I, ¹³¹I, ³²P or ³³P labelled ascorbic acids could beused in the treatment of metastatic bone disease. In such applicationsthe therapeutic effect would be due to the local targeted radioactivedose delivered to specific cells, as opposed to any pharmacologicaleffect due to the ascorbic acid. Whichever detectable moiety is chosen,it is strongly preferred that it is bound to the ascorbic acid in such away that it does not undergo facile metabolism (either in vivo or invitro), since such metabolism would result in the biodistribution of thedetectable moiety no longer reflecting that of the ascorbic acid.

When the detectable moiety is a hyperpolarised ¹³C atom, this atom mayform an integral part of the chemical structure of the ascorbic acid, orcan be attached as a supplemental group. Most other detectable moietiesmust form supplemental structural elements, and can be attached at the2, 3, 4, 5 or 6-positions of the ascorbic acid derivative. Preferredpositions for the detectable moiety are the 2, 3 and 6-positions, withthe 6-position being most preferred.

By the term ‘protecting group’ is meant those moieties known to thoseskilled in the art which would prevent metabolic modification of theascorbic acid moiety. This may include alkyl, alkoxyalkyl, benzyl oracyl groups. The protecting group may also function to prevent anyoxidation or other chemical degradation process of the ascorbic acidhydroxyl groups, and for such purposes is chosen to be sufficientlylabile so that the protecting group is cleaved during the labelling orradiolabelling process.

When the detectable moiety is a radioactive or paramagnetic metal, themetal ion is always complexed. This metal complex is preferably achievedby attaching a ligand which binds strongly to metals to the ascorbicacid moiety. Such strongly metal-binding ligands include monodentatecompounds which bind well to transition metals such as phosphines,isonitriles or hydrazides, and polydentates such as chelating agents.The ligand-ascorbic acid conjugate is complexed with the radioactive orparamagnetic metal ion, and the metal binds selectively to the ligand,giving a metal complex of the ligand linked to the ascorbic acidderivative.

The chelating agents of the present invention comprise 2-10 metal donoratoms covalently linked together by a non-coordinating backbone.Suitable bidentate chelating agents include bisphosphonates anddiphosphines. Bisphosphonate complexes of radiometals have the advantagethat the radiometal complex is already targeted to the bone in vivo.Preferred chelating agents have 4-8 metal donor atoms and have the metaldonor atoms in either an open chain or macrocyclic arrangement orcombinations thereof. Most preferred chelating agents have 4-6 metaldonor atoms and form 5- or 6-membered chelate rings when coordinated tothe metal centre. Such polydentate and/or macrocyclic chelating agentsform stable metal complexes which can survive challenge by endogenouscompeting ligands for the metal in vivo such as transferrin or plasmaproteins. The metal complex should also preferably be of lowlipophilicity (since high lipophilicity is often related to non-specificuptake), and exhibit low plasma protein binding since plasma bound labelagain contributes to undesirable high, non-specific background for theimaging agent.

Examples of suitable chelating agents are diaminedioximes (U.S. Pat. No.4,615,876) or such chelates incorporating amide donors (WO 94/08949);the tetradentate chelates of WO 94/22816; N₂S₂ diaminedithiols,diamidedithiols or amideaminedithiols; N₃S thioltriamides; N₂O₂diaminediphenols; N₄ chelates such as tetraamines, macrocyclic amines oramide chelates such as cyclam, oxocyclam (which forms a neutraltechnetium complex) or dioxocyclam; or dithiosemicarbazones. The abovedescribed chelates are particularly suitable for technetium, but areuseful for other metals also. Other suitable chelates are described inWO 91/01144, which includes chelates which are particularly suitable forindium, yttrium and gadolinium, especially macrocyclic aminocarboxylateand aminophosphonic acid chelates. Chelates which form non-ionic (i.e.neutral) metal complexes of gadolinium are known and described in U.S.Pat. No. 4,885,363. The chelate may also comprise a short sequence ofamino acids such as the Cys/aminoacid/Cys tripeptide of WO 92/13572 orthe peptide chelates described in EP 0719790 A2.

When the detectable moiety is a radioactive isotope of iodine, theradioiodine atom is preferably attached via a direct covalent bond to anaromatic ring, such as a benzene ring, or to a vinyl group, since it isknown that iodine atoms bound to saturated aliphatic systems are proneto in vivo metabolism and hence loss of the detectable moiety.

Bone metastases may be osteoblastic (10%), osteolytic (65%) or mixed(25%) in appearance. It is believed that the compounds of the presentinvention are only taken up by activated osteoblasts at sites ofincreased bone turnover, i.e. active lesions. Such sites include areasof hyperproliferation associated with metastatic bone disease, as wellas other bone pathologies. The present compounds are therefore expectedto be of high diagnostic and prognostic value for osteoblastic lesions,and to allow rapid monitoring of disease and treatment progress. This isin contrast to prior art [^(99m)Tc]-MDP, which accumulates at sites ofcollagen production long after tumour cells are dead, giving noinformation about cell viability. The compounds of the present inventionmay also help to prevent the occurrence of false negative scans, via thevisualisation of osteolytic lesions due to associated osteoblasticactivity and allow early diagnosis of small lesions due to the specificuptake mechanism. The relatively rapid expected clearance time shouldallow rapid imaging and high patient throughput. The present compoundsmay also accumulate in fibroblasts and could hence be useful in thediagnostic imaging of sites of wound repair, and may also be useful inthe diagnosis of other bone pathologies, for example osteoporosis andarthritis.

A further aspect of the present invention is the disclosure of novelascorbic acid derivatives. These may be useful as pharmaceuticals forthe treatment of tumours known to accumulate ascorbic acid, inparticular bone tumours, and may also be attached to therapeuticradioisotopes or cytotoxic drugs.

Novel ascorbic acid derivatives, including those labelled withnon-radioactive ¹²⁷I, have been prepared, and shown to compete with¹⁴C-labelled ascorbic acid for uptake into murine pre-osteoblast(MC3T3-E1) cells. Such derivatives have essentially identical chemicalproperties to the radioactive counterparts labelled with radioiodinee.g. ¹²³I or ¹³¹I. In addition, a few known compounds have also beensynthesised and shown to compete with ¹⁴C-labelled ascorbic acid foruptake into MC3T3-E1 cells. Furthermore, a ¹⁴C-labelled ascorbic acidderivative (compound 17) has been shown to accumulate in primary ratosteoblasts over a 5 hour period and remains stable over this time.Using autoradiography, accumulation has also been demonstrated inmineralised bone nodules produced by rat osteoblasts over a 21-dayculture period. In vivo, the amount of ¹⁴C-compound 17 accumulating inthe epiphysis of the rat 60 minutes after i.v. injection was compared tothat accumulating in the diaphysis and was found to be 2.3-fold greaterdue to increased osteoblast activity at these sites.

The compounds of the present invention may be prepared as follows. Whenthe detectable moiety is radioactive iodine, the substituent linked toascorbic acid must include a non-radioactive halogen atom (to permitradioiodine exchange), an activated aromatic ring (e.g. a phenol group),an organometallic precursor compound such as a trialkyltin ortrialkylsilyl, an organic precursor such as triazenes or other suchmoiety known to those skilled in the art. Examples of suitablesubstituents to which radioactive iodine can be attached are givenbelow:

Both substituents contain groups which permit facile radioiodinesubstitution onto the aromatic ring. Alternative substituents containingradioactive iodine can be synthesised by direct iodination viaradiohalogen exchange, e.g.

Groups for substitution of radioiodine can be attached to ascorbic acidas follows. A substituent functionalised with a carboxylic acid groupcan be reacted with the 6-OH of ascorbic acid to give an ester link [J.Carbohyd. Chem. 17(3) 397-404 (1998)]. Alternatively6-bromo-6-deoxy-L-ascorbic acid [Suskovic, Croat. Chem. Acta, 58, 231(1985)] can be reacted with an amino or thiol-functionalised substituentto give an amino [Kralj et al, Eur. J. Med. Chem., 31, 23, (1996)] orthioether [Carbohyd. Res., 134, 321, (1984)] link.6-Amino-6-deoxy-L-ascorbic acid [Suskovic, Croat. Chem. Acta, 62, 537(1989)] can be reacted with substituents functionalised with acarboxylic acid or an active ester to give an amide link. Personsskilled in the art will recognise that many alternative syntheses ofascorbic acid derivatives suitable for radioiodination are possiblebased on this disclosure.

An alternative method for synthesising ascorbic acid derivativessuitable for radioiodination involves rearrangement of a L-gulonate(shown below) under acidic conditions [Crawford et at, Adv. Carbohyd.Chem Biochem., 37, 79 (1980)].

The 4,6-isopropylidene protecting group is removed and a substituentsuitable for radioiodination is linked to the primary hydroxyl of theL-gulonate via an ether or ester link, for example, and the modifiedL-gulonate rearranged to give the corresponding ascorbic acidderivative. Alternatively the primary hydroxyl can be substituted for abromo or amino group for reaction with an appropriately functionalisedgroup suitable for radioiodination, prior to rearrangement to theascorbic acid.

When the detectable moiety is a radioactive or paramagnetic metal ion, achelating agent is attached to the ascorbate giving a chelate-ascorbicacid conjugate. Such chelate-ascorbic acid conjugates can be preparedusing the bifunctional chelate approach. Thus, it is well known toprepare chelating agents which have attached thereto a functional group(“bifunctional chelates”). Functional groups that have been attached tochelating agents include: amine, thiocyanate, maleimide and active estersuch as N-hydroxysuccinimide. Such bifunctional chelates can be reactedwith suitable functional groups on the ascorbic acid to form the desiredconjugate. Examples of chelate-amine conjugates for diaminedioximeligands are given in WO 95/19187. In the particular case of ascorbicacid, a chelating agent can be attached at the 6-position as follows.Ascorbic acid can be reacted with a chelate-carboxylic acid conjugate togive a chelate-ascorbic acid derivative linked via an ester bond.6-COOH-6-deoxy-L-ascorbic acid [Stuber et al, Carbohyd. Res., 60, 25(1978)] can be reacted with a chelate-amine conjugate, or6-NH₂6-deoxy-L-ascorbic acid reacted with a chelate-active ester orchelate-carboxylic acid conjugate to give chelate-ascorbic acidderivatives linked via amide bonds. 6-Br6-deoxy-L-ascorbic acid can bereacted with a chelate-amine or chelate-thiol conjugate to give eitheran amine or thioether link.

An alternative method for synthesising ascorbic acid derivativesinvolves rearrangement of an L-gulonate derivative as described above.This reaction can be used in the synthesis of chelate-ascorbic acidconjugates. A chelate can be linked to the L-gulonate using one of themethods described above and the resulting chelate-L-gulonate conjugaterearranged to give the corresponding chelate-ascorbic acid derivative.Persons skilled in the art will recognise that many alternativesyntheses of chelate-ascorbic acid conjugates are possible based on thisdisclosure.

When the detectable moiety is a hyperpolarised atom, such as ahyperpolarised ¹³C atom, the desired hyperpolarised compound can beprepared by polarisation exchange from a hyperpolarised gas (such as¹²⁹Xe or ³He) to a suitable ¹³C-enriched ascorbic acid derivative. Both[1-¹³C]- and [2-¹³C]-labelled ascorbic acid derivatives are known, andhave been used to examine transport and redox cycling in humanerythrocytes [Himmelreich et al. Biochem. 37, 7578 (1998)]. ¹³C-enrichedascorbic acid derivatives can also be prepared in an analogous manner tothe literature synthetic routes for ¹⁴C-labelled ascorbic acidderivatives. Thus, Hornig et al [Int. J. Vit. Nutr. Res. 42, 223 (1972)and ibid 42, 511 (1972)] have studied the autoradiographicbiodistribution of [1-¹⁴C]-L-ascorbic acid in normally fed and vitamin Cdeficient guinea pigs following intravenous injection. Karr et al [J.Lab. Comp., 6, 155 (1970)] have also prepared [6-¹⁴C]-L-ascorbic acidand [5-¹⁴C]-L-ascorbic acid from D-glucose-1-¹⁴C and D-glucose2-¹⁴C,respectively. Williams et al [Carbohyd. Res., 63, 149 (1978)] describethe synthesis of [4-¹⁴C]-L-ascorbic acid from D-[3-¹⁴C]glucopyranose and[6-¹⁴C]-L-ascorbic acid from D-[1-¹⁴C]glucopyranose.

Unlabelled ascorbic acid derivatives of the present invention have beentested for their ability to compete for uptake of ¹⁴C-ascorbic acid intoMC3T3-E1 cells, a murine pre-osteoblast cell line. MC3T3-E1 cells aregrown in tissue culture plates and the appropriate assay solutioncontaining a standard concentration of ¹⁴C-ascorbic acid plus acompeting concentration of ascorbic acid derivative added to each well.The amount of ¹⁴C-ascorbic acid taken up by the cells in 60 min is thenmeasured.

Of compounds 11 to 15, only those containing an iodophenyl, bromophenylor iodovinyl substituent were found to compete for uptake of¹⁴C-ascorbic acid into MC3T3-E1 cells. Results are given in Table 10.Both Compounds 16 and 17 competed for uptake of ¹⁴C-ascorbic acid intoMC3T3-E1 cells, although competition by compound 16 was very weak.Although both these compounds are known, neither has been reported inthe literature to show competition with ¹⁴C-ascorbic acid for uptakeinto pre-osteoblast or osteoblast cells.

The present invention also relates to kits for the preparation ofascorbic acid derivatives labelled with a detectable moiety. The kitsare designed to give sterile products suitable for human administration,e.g. via injection into the bloodstream. Possible embodiments arediscussed below. When the detectable moiety is ^(99m)Tc, the kit wouldcomprise a vial containing either an ascorbic acid derivative suitablefor forming a metal complex with ^(99m)Tc or a chelate-ascorbic acidconjugate, together with a pharmaceutically acceptable reducing agentsuch as sodium dithionite, sodium bisulphite, formamidine sulphonicacid, stannous ion, Fe(II) or Cu(I). The reducing agent is preferably astannous salt such as stannous chloride or stannous tartrate.

Alternatively, the ascorbic acid derivative or chelating agent-ascorbicacid conjugate could be present as the metal complex of a suitablenon-radioactive metal, which, upon addition of the radiometal, undergoestransmetallation (i.e. ligand exchange) giving the desired product. Thekit is preferably lyophilised and is designed to be reconstituted withsterile ^(99m)Tc-pertechnetate (TcO₄ ⁻) from a ^(99m)Tc radioisotopegenerator to give a solution suitable for human administration withoutfurther manipulation.

The agents for the present invention may also be provided in a unit doseform ready for human injection and could for example be supplied in apre-filled sterile syringe. When the detectable moiety is a radioactiveisotope such as ^(99m)Tc, the syringe containing the unit dose wouldalso be supplied with a syringe shield (to protect the operator frompotential radioactive dose).

The above kits or pre-filled syringes may optionally contain furtheringredients such as buffers; pharmaceutically acceptable solubilisers(e.g. cyclodextrins or surfactants such as Pluronic, Tween orphospholipids); pharmaceutically acceptable stabilisers/antioxidants(such as gentisic acid or para-aminobenzoic acid) or bulking agents forlyophilisation (such as sodium chloride or mannitol).

The structure of compound 10 is given in Scheme 1. The structures ofcompounds 11 to 28 are given in Tables 1 to 4. The preparation ofcompounds 10-19 and 21-28 is described in Examples 1 to 6. NMR data forthe compounds is given in Tables 4 to 9. The biological properties ofCompounds 11 to 18 are shown in Example 7 and Table 10. The biologicalproperties of Compound 17 are further discussed in Examples 8 and 9.

TABLE 1

Compound n R² R³ R⁴ 11 0 OCH₃ OH H 12 0 H I H 13 0 H Br H 14 1 H I H

TABLE 2

Compound Number R⁵ 15 1-Iodovinyl

TABLE 3 Compounds 16 and 17 are literature compounds.

Compound number R⁶ 16 Ph— 17 PhCH₂— 18 Ph(2-COOH)— 19 HO₂C(CH₂)₂— 20(4-MeOPh)CH₂NH(CO)CH₂— 21 [Diaminediphenol]-linker1- 22[Diaminediphenol]-linker2- 23 [Pn216]-linker1 24 [Pn216]-linker2 25[Isopropylamine]-linker1 26 [Isopropylamine]-linker2 27 [Hynic]-linker2

where: [Diaminediphenol]-linkers- are:

where: [Pn216]-linkers- are:

where: [Isopropylamine]-linkers- are:

where: [Hynic]-linker- is:

TABLE 4

Compound number R⁷ 28 MAG3 note: the full structure of Compound 28 isgiven in Table 9.

EXPERIMENTAL EXAMPLE 1 Synthesis of a Bisphosphonate Conjugate 10

¹H NMR chemical shifts are with respect to TMS; ¹³C NMR chemical shiftsare with respect to CDCl₃ at 77 p.p.m. or, for aqueous solutions, MeOHat 49.2 p.p.m.; ³¹P NMR chemical shifts are with respect to external 85%H₃PO₄.

3,3-Bis(diisopropoxyphosphinyl)propionic acid (3)

Sodium hydride (700 mg, 0.29 mmol) was added in small portions under astream of dry nitrogen to a vigorously stirred solution oftetraisopropyl methane-1,1-bisphosphonate (1) (5.0 gm, 14.5 mmol) in drytoluene (25 ml). After the effervescence had stopped, stirring wascontinued for 15 minutes. Ethylbromoacetate (3.2 gm, 19.2 mmol) was thenadded dropwise over a period (2 min) whereupon the flask became warm anda white precipitate began to form. Stirring was continued for a further2 hours at room temperature. Water (20 ml) was added then carefully andthe mixture vigorously stirred. The toluene layer was separated andextracted with water (20 ml). The aqueous extracts were combined, washedwith ether (50 ml), acidified to pH 1 and re-extracted withdichloromethane (2×25 ml). The dichloromethane extracts were combined,dried (MgSO₄), filtered and solvent evaporated under reduced pressure toleave the acid (3) as a pale yellow liquid (800 mg). The toluene layerwas also dried (MgSO₄), filtered, and solvent evaporated to leave an oilwhich was found to be the ethyl ester (2) and unreacted startingmaterial. This oil was dissolved in a methanol:water mixture (20 ml,3:1) containing lithium hydroxide (1 g, 24 mmol) and the solutionstirred at room temperature for 16 h. The methanol was removed byevaporation and water (20 ml) was then added. This aqueous solution waswashed with ether (2×20 ml), acidified to pH 1 with dilute HCl and thenextracted with dichloromethane (2×25 ml). The dichloromethane extractswere combined, dried (MgSO₄), filtered and volatile components removedunder reduced pressure to leave the acid (3) (2.77 g) as a pale yellowliquid. The combined product (3) (3.57 g, 61%) was sufficiently pure tobe used in subsequent reactions without further purification.

δ_(P)(CDCl₃) 21.79

δ_(C)(CDCl₃) 23.7 (m), 30.6 (s), 34.0 (t, J_(PC)=138 Hz), 71.8 (m),172.8 (s)

(N-Succinimidyl) 3,3-Bis(bisisopropoxyphosphinyl)Propionate (4)

A solution of dicyclohexylcarbodiimide (2.0 g, 9.71 mmol) indichloromethane (10 ml) was added in one portion to a stirred solutionof 3,3-bis(bisisopropoxy-phosphinyl)propionic acid (3) (3.57 g, 8.88mmol) and N-hydroxysuccinimide (1.15 g, 10 mmol) in dry dichloromethane(35 ml). After about 10 minutes a white precipitate of dicyclohexylureabegan to appear. The mixture was stirred for 16 h and the solid formedwas filtered off and washed with dichloromethane (15 ml). The solventwas removed under reduced pressure from the combined dichloromethanesolutions to leave a viscous residue, which was shown by NMR to be thetitle compound in a good state of purity. Final purification of thisresidue was carried out using chromatography on silica with adichloromethane:methanol mixture (19:1) as eluant. The product (4.0 g,91%) (R_(f)0.2) was isolated as a viscous oil.

δ_(P)(CDCl₃) 20.0

δ_(H)(CDCl₃) 1.25-1.31 (24H, m, CH₃×8), 2.77 (4H, s, CH₂×2), 2.83 (1H,m, CH), 2.7-3.1 (2H, m, CH₂), 4.37 (4H, dq, J_(HH)=6 Hz, J_(PH)=13.5 Hz,CH×4)

(R)-5-(2-azido-(S)-1-hydroxyethyl)-3,4-dibenzyloxy-5H-furan-2-one (6)

(R)-5-[2-(4-methylphenylsulfonyloxy)-(S)-1-hydroxyethyl]-3,4dibenzyloxy-5H-furan-2-one(5)^(†) (550 mg, 1.1 mmol), sodium azide (190 mg, 1.7 mmol) and methanol(2 ml) were heated under reflux for 6 hours. The reaction mixture wascooled and most of the solvent removed by evaporation at roomtemperature. The resulting material was partitioned between water (25ml) and dichloromethane (25 ml) and the aqueous phase extracted withdichloromethane (25 ml). The combined organic fractions were dried(MgSO₄), filtered and volatile components evaporated under reducedpressure (8 mm/Hg) at room temperature to give the product (6) as ayellow waxy solid (375 mg, 89%). This material was used without furtherpurification.

δ_(H)(CDCl₃) 3.22 (1H, dd, J=6 and 12.5 Hz, CH₂N₃), 3.40 (1H, br d, OH),3.46 (1H, dd, J=7 and 12.5 Hz, CH₂N₃), 3.88 (1H, br m, CHO), 4.55 (1H,d, J=2 Hz , CHO ring), 4.95 (2H, br s, OCH₂), 5.03 (1H, d, J=12 Hz,OCH), 5.08 (1H, d, J=12 Hz, CHO), 7.12 (2H, m, Ar), 7.21-7.31 (8H, m,Ar)

† V. F. Dallacker and J. Sanders, Chem. Zeit., 1985, 109, 197-202

(R)-5-(2-amino-(S)-1-hydroxyethyl)-3,4-dibenzyloxy-5H-furan-2-one (7)

Triphenylphosphine (600 mg, 2.4 mmol) was added to a stirred solution of(R)-5-(2-azido-(S)-1-hydroxyethyl)-3,4-dibenzyloxy-5H-furan-2-one (5)(750 mg, 2 mmol) in THF (10 ml) and after a few minutes gas was evolved.The stirring was continued until the effervescence had ceased (typically2-3 h) and water (2 ml) was then added and the mixture stirred for afurther 1 hour. The THF was removed under reduced pressure and theresidue partitioned between dichloromethane (25 ml) and water (25 ml).The organic phase was separated, dried (MgSO₄), filtered and anyvolatile components evaporated under reduced pressure (45° C. at 8mm/Hg) to leave a viscous orange residue which was purified bychromatography on silica. Initial elution with adichloromethane:methanol (19:1) mixture removed the less polarby-products and the product was then isolated by elution with adichloromethane:methanol (3:1) mixture as a pale yellow oil (150 mg;21%) (R_(f)0.25).

δ_(H)(CDCl₃) 2.49 (3H, br s, NH₂ and OH), 2.76 (1H, dd, J=5 and 13 Hz,NCH), 2.83 (1H, dd, J=7 and 13 Hz, NCH), 3.70 (1H, m, CHO), 4.47 (1H, d,J=2 Hz, CHO ring), 4.98 (2H, s, OCH₂), 5.05 (1H, d, J=12 Hz, OCH), 5.11(1H, d, J=12 Hz, CHO), 7.10-7.17 (2H, m, Ar), 7.20-7.32 (8H, m, Ar)

(R)-5-(3-aza-6.6-bis(bisisopropoxyphosphinyl)-(S)-1-hydroxy-oxo-hexyl)-3,4-dibenzyloxy-5H-furan-2-one(8)

(R)-5-(2-amino-(S)-1-hydroxyethyl)-3,4-dibenzyloxy-5H-furan-2-one (7)(180 mg, 0.5 mmol) in dry dichloromethane (1 ml) was added in oneportion to a stirred solution of (N-succinimidyl)3,3-bis(bisisopropoxyphosphinyl)propionate (4) (250 mg, 0.5 mmol) in drydichloromethane (1 ml), whereupon crystals of N-hydroxysuccinimide beganto precipitate. The mixture was stirred for 30 min, dichloromethane (10ml) was then added and the solution washed with water (2×10 ml). Theorganic phase was then dried (MgSO₄), filtered and solvent evaporatedunder reduced pressure to leave (8) as a pale yellow viscous oil in avirtually pure state. Final purification could be achieved bychromatography on silica with a dichloromethane:methanol mixture (19:1)as the eluant. The product (8) (180 mg, 49%) (R_(f)0.25) was obtained asa pale yellow viscous oil.

δ_(P)(CDCl₃) 21.9 (d, J_(PP)=4 Hz), 22.0 (d, J_(PP)=4 Hz)

Mass Spec (FABS), Calculated for C₃₅H₅₂NO₁₂P₂ 740.2965 (M+H)⁺, found740.2965.

(R)-5-(3-aza-6,6-bis(bisisopropoxyphosphinyl)-(S)-1-hydroxy-4-oxo-hexyl)-3,4-dihydroxy-5H-furan-2-one(9)

A solution of(R)-5-(3-aza-6,6-bis(bisisopropoxyphosphinyl)-(S)-1-hydroxy-4-oxo-hexyl)-3,4-dibenzyloxy-5H-furan-2-one(8) (700 mg, 0.96 mmol) in methanol (10 ml) was hydrogenated over apalladium catalyst (200 mg, 10% Pd/C) and in a hydrogen atmosphere (30atm) for 2 hours at room temperature. The catalyst was removed byfiltration through Celite and the filter cake washed with methanol (50ml). These washings were combined with the methanol filtrate and thevolatile components evaporated under reduced pressure to leave (9) as aviscous oil in a good state of purity. The residue was purified bychromatography on silica using an ethyl acetate;methanol (9:1) mixtureas the eluant. The pure product (9) (50 mg, 9%) (R_(f)0.3) was isolatedas a cream coloured solid.

δ_(P)(CDCl₃) 21.8 (br s), 22.98 (br s)

Mass Spec (FABS), Calculated for C₂₁H₄₀NO₁₂P₂ 560.2026 (M+H)⁺, found560.2026.

6-[(R)-5-(2,5-dihydro-3,4-dihydroxy-2-oxo-furan-5-yl)]-4-aza-(S)-6-hydroxy-3-oxo-hexane-1,1-bisphosphonicacid (10)

To a solution of(R)-5-(3-aza-6,6-bis(bisisopropoxyphosphinyl)-(S)-1-hydroxy4-oxo-hexyl)-3,4-dihydroxy-5H-furan-2-one(9) (370 mg, 0.6 mmol) in dichloromethane (10 ml) was addedbromotrimethylsilane (1 g, 6.4 mmol). The mixture was heated underreflux for 6 h and the solvent was then removed under reduced pressure.Methanol (25 ml) was added and volatile components evaporated underreduced pressure. This process was repeated twice to leave a brownresidue. The product (10) was purified by reverse phase HPLC on a C₁₈column using aqueous methanol (50%) as eluant and isolated as a palebrown solid (120 mg; 46%).

δ_(P)(D₂O) 21 (br)

δ_(C)(D₂O) 32.1 (br s), 34.8 (t, J_(PC)=138 Hz), 42.1 (s), 67.1 (s),76.8 (s), 118.1 (s), 155.5 (s), 173.4 (s), 173.5 (br s).

EXAMPLE 2 Synthesis of Compounds 11 to 15

Compounds 11 to 15, all esters of ascorbic acid, were synthesiseddirectly from ascorbic acid and the corresponding carboxylic acid usingthe method reported by Gan et al [J. Carbohyd. Chem., 17, 397-404(1998)]. Compounds 11 and 13 to 15 were all synthesised using a similarprocedure to that given below for 6-O-(4-iodobenzoyl)-L-ascorbic acid(Compound 12):

4-Iodobenzoic acid (0.5 g, 2.48 mmol), and L-ascorbic acid (2 g, 11.3mmol) were mixed and stirred in concentrated sulphuric acid (10 ml) for24 hrs at room temperature. The reaction was quenched by the addition ofice and solid sodium chloride. The mixture was extracted with ethylacetate and the organic layer dried over magnesium sulphate. The titlecompound was isolated by evaporation under reduced pressure followed bycrystallisation from a chloroform/hexane mixture and drying under vacuum(200 mg, 5.6 mmol).

EXAMPLE 3 Synthesis of Compounds 16 and 17

These compounds were synthesised using methods similar to the literatureprocedures, [Carbohyd. Res., 134, 321, (1984)] for compound 16 and [J.Biol. Chem., 271, 26032, (1996)] for compound 17. ¹⁴C-compound 17 wasprepared using a similar method but starting with ¹⁴C-bromo ascorbicacid.

EXAMPLE 4 Synthesis of Compounds 18 and 19

These compounds were synthesised using very similar methods. To asuspension of sodium carbonate (960 mg) in methanol (2 ml) and water (6ml) was added 6-bromo-L-ascorbic acid (500 mg) and thiosalicyclic acid(350 mg; for compound 18) or 3-mercaptoproprionic acid (240 mg; forcompound 19). The resulting mixture was stirred for at least 4 hr atroom temperature and the reaction checked for completion by TLC. Thereaction was acidified using 2M HCl and the product extracted into ethylacetate. The organic layer was washed with brine and dried over MgSO₄.Filtration and evaporation to dryness yielded a buff-coloured solidwhich was treated with chloroform and filtered to yield 590 mg compound18/200 mg compound 19.

EXAMPLE 5 Synthesis of Compounds 21-27

Compound 21

The trityl protected acid was prepared by a method described in, [Inorg.Chem 23 (23) 3795-3797 (1984)]. S-trityl mercapto acetic acid (5.6 g)was dissolved in dry acetonitrile (60 ml) under nitrogen. To this wasadded a solution of N-hydroxysuccinimide (2 g) in acetonitrile (10 ml).This was followed by the addition of DCC (4.4 g) in acetonitrile (20ml). The mixture was stirred overnight at room temperature. The productwas isolated by filtration followed by evaporation of the filtrate togive a white solid (6 g). The S-trityl-mercaptoacetic acid NHS ester(400 mg) was dissolved in dry dichloromethane (10 ml) under nitrogen. Tothis was added the diaminodiphenol ligand (300 mg), followed bytriethylamine (146 μl). The reaction was stirred at room temperatureovernight and the product isolated by evaporation followed by flashcolumn chromatography on silica. The trityl protectedDiaminodiphenol-linker (270 mg) was dissolved in DCM (6 ml) to this wasadded triethylsilane (100 μl) followed by trifluoroacetic acid (300 μl).The mixture was stirred for 4 hrs at room temperature, after which itwas evaporated to dryness and used directly in the final step, which wascarried out as described for compounds 18 and 19.

Compounds 22-27 were prepared in an analogous manner.

EXAMPLE 6 Synthesis of Compound 28

This compound was synthesised using a combination of methods describedin the literature. The chelate MAG3 was prepared using the methoddescribed by Winnard et al [Nucl Med Biol 24 425-432 (1997)].N-methyl-6-amino-6deoxy-O³-benzyl-L-ascorbic acid was prepared by amethod described by Kralj M et al [Eur J Med Chem 31 23-35 (1996). Thecoupling reaction between MAG3 and theN-methyl6-amino-6-deoxy-O³-benzyl-L-ascorbic acid was achieved usingPyBrop (an agent specifically used for N-methyl amines) in a mannersimilar to that described by Coste J [Tet. Lett. 32 (17) 1967-1970(1991). Thus, to N-methyl-6-amino-6-deoxy-O³-benzyl-L-ascorbic acid (91mg, 3.26×10⁻⁴ moles) in dry DMF (5 ml) under nitrogen was added MAG3(100 mg, 3.26×10⁻⁴ moles). While stirring at room temperature,diisopropylethylamine (211 mg, 1.63×10⁻³ mol) was added followed byPyBrop (182.5 mg, 3.914×10⁻⁴ mol). After stirring overnight the solventwas removed in vacuo and the product isolated by preparative HPLC.

TABLE 5 ¹H NMR Data for compounds 11, 12, 13 and 14.

NMR data Compound Compound Compound Compound (ppm) 11 12 13 14 R² 3.8  —— — R³ — — — — R⁴ — — — — (CH₂)n — — — 3.57 H5 3.77 4.85 4.07 3.98H6_(b) 4.12 4.45 4.20 4.12 H6_(a) 4.32 4.65 4.28 4.18 H4 4.72 4.85 4.664.56 Aryl 6.6-7.5 8.0-7.5 7.7-7.0 7.5-7.05

TABLE 6 ¹H NMR Data for compound 15.

NMR data (ppm) Compound 15 H-5 3.7 H-6_(a,b) 4.2 H-4 4.28 I-vinyl_(a)6.83 I-vinyl_(b) 6.22

TABLE 7 ¹H NMR Data for Compounds 16 and 17.

NMR data (ppm) Compound 17 Compound 16 CH₂ 2.70 CH₂(H-6_(a,b)) 3.673.22-3.26 CH(H-5) 3.83 3.95-3.99 CH(H-4) 4.35 4.88 Aryl 7.13-7.187.20-7.44

TABLE 8 ¹H NMR Data for compounds 21-27 Cmpd number ppm ppm ppm ppm Ppmppm ppm ppm 21 1.2 2.6 3.0 3.5 4.0 4.6 4.8 6.6-7.3 s,3-H d,2-H s,4-Hm,4-H m,1-H m,4-H d,1-H m,8-H 22 0.7 2.1 2.5 2.9 3.3-3.4 3.8 4.3 6.3-7.3s,3-H m,2-H m,4-H s,2-H m,3-H s,4-H d,1-H m,12-H 23 1.5 1.9 2.2-2.93.0-3.1 3.2-3.3 3.3-3.4 4.0 4.8 s,12-H s,6-H m,4-H m,4-H m,4-H m,4-Hm,1-H d,1-H 24 1.5 1.8 2.5-3.0 3.5 3.8 4.0 4.5 7.4-7.9 s,12-H s,6-Hm,12-H s,1-H s,1-H m,1-H s,1-H m,4-H 25 0.7 2.4 2.8 3.6 4.3 d,6-H d,2-Hd,2-H m,2-H d,1-H 26 1.3 2.2 3.7 3.8 4.1 4.4 7.4 7.6 d,6-H d,2-H d,2-Hm,1-H m,1-H d,1-H d,2-H d,2-H 27 1.8 2.7 3.5 3.9 4.8 7.0 8.2 8.4 s,9-Hm,4-H t,2-H m,1-H d,1-H d,1-H dd,1-H d,1-H

TABLE 9 ¹H NMR Data for compound 28

NMR data(ppm) Compound 28 SCOCH3 2.3 NCH3 3.0 SCH2 3.3 CH2(H-6_(a,b))3.4-3.6 Amide-CH2 3.6 Amide CH2x2 3.9 CH(H-5) 4.1 CH(H-4) 4.6 Benzyl-CH25.5 Aryl 7.4

EXAMPLE 7 Standard Cell Uptake Assay

Solutions and Reagents

Transport buffer: 134 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄,20 mM HEPES, 10 mM glucose, pH7.3, stored at room temperature

Assay buffer: Transport buffer+40 μM homocysteine ¹⁴C-ascorbic acid(Amersham Pharmacia Biotech)

Stock solution: 50 μCi in 50 μl Analar water, stored at −20° C.

Assay solution: 200 nCi ¹⁴C-ascorbic acid in 200 μl assay buffer/well(125 μM)

Ascorbic acid (Sigma)

Dehydroascorbic acid (Sigma)

Test Compounds

Stop buffer: 200 μM phloretin in assay buffer, chilled to 4° C.

Lysis solution: 0.1% SDS in Analar water

Method

MC3T3-E1 murine pre-osteoblast cells were seeded in a 24-well plate at2×10⁵ cells/well in 1 ml minimal essential media plus 10% FCS,penicillin and streptomycin. Cells were grown overnight at 37° C., 5%CO₂. Assay buffer and solutions were prepared on the morning of theassay. Cells were examined under the microscope for confluence andviability. Growth media was removed using a Liquipippette and each wellwashed with 2×200 μl assay buffer. The appropriate assay solution wasadded to each well and the plate incubated at 37° C. for the requiredtime. To stop the reaction, 0.5 ml chilled stop buffer was added to eachwell. Each well was then washed with 0.5 ml stop buffer. Cells werelysed in 0.1% SDS solution for approximately 10 min and solutionstransferred to scintillation vials. Each well was then washed with assaybuffer and this wash solution also transferred to the appropriatescintillation vial. Scintillation fluid (5 ml) was added to each vial,the vials vortexed and counted on a Rackbeta counter.

TABLE 10 Results of cell assay Compound Competition IC50 (μM) Ascorbicacid Yes 258 11 No — 12 Yes 384 13 Yes 279 14 Yes 1880  15 Yes 796 16Yes 1847  17 Yes 108 18 No — 19 Not tested —

EXAMPLE 8 Uptake of ¹⁴C-Compound 17 into Rat Osteoblasts

Preparation and Culture of Primary Rat Osteoblasts

Foetus heads were collected and sprayed with 70% isopropanol. Calvariawere then removed by cutting the skin from the top of the head, makingone incision through the calvaria from the external auditory meatusacross behind the parietal bones and a second forward to above the eyesockets. When all the calvaria were isolated, they were rinsed with PBS(Ca²⁺ and Mg²⁺-free), then incubated in trypsin at 37° C. for 10minutes. Calvaria were transferred into 0.2% collagenase (type II) inHBSS for 30 minutes at 37° C. This collagenase digestion was repeatedfor 60 minutes to release the osteoblast populations. Supernatant wasremoved and spun down to obtain a cell pellet. The pellet wasresuspended in media, viable cells counted and plated out into a T75flask. Once confluent, cells were subcultured into 6-multiwell plates at2×10⁴-2×10⁵ cells/well in 3 ml medium containing 1 mMβ-glycerophosphate. In addition ascorbic acid (50 μg/ml), ¹⁴C-ascorbicacid (19.6 μCi/ml) or ¹⁴C-compound 7 (2.8 μCi/ml) were incubated withosteoblasts over the culture period. Cells were fed two to three timesper week and cultured for up to 21 days. Nodules became macroscopicallyvisible at around 7-10 days and commenced mineralisation (assessed byAlizarin red staining) soon after.

Autoradiography

At the end of the culture period, cells were fixed with 2.5%glutaraldehyde and dehydrated with an increasing series of ethanolconcentrations. Approximately 2 ml of hypercoat emulsion (LM-1; APB) wasadded to each well and incubated at room temperature in the dark for24-36 hours. The emulsion was then developed following standard methods.

HPLC Analysis

The separation of DHAA, M, compound 7 and Benzyl Mercaptan wassuccessfully completed using the following HPLC profile:

Mobile phase: A and B=50% acetonitrile:50% 50 mM KH₂PO₄

Detector: UV (235 nm)/Flow Scintillation Analyser (β-radiation)

Flow rate: 1 ml/min

Column: Waters Spherisorb S5NH2—5 μm—4.6×250 mm

(y = mV.min & x = nmoles) Retention Time (min) Calibration Graph DHAA3.34 ± 0.10% y = 121946 nmoles AA 5.74 ± 0.20% y = 215320 nmolesCompound 7 3.71 ± 0.27% y = 255088 nmoles Benzyl Mercaptan 2.44 ± 0.14%y = 78710 nmoles

¹⁴C-compound 7 samples analysed using the HPLC profile above were:

MEM solution (standard)

0.1%SDS solution (standard)

MEM-Removed from cells

0.1% SDS - Lysed cells, at time points of 1, 3, 5, 24, 48, 72, 96 and168 hrs.

Results

The HPLC results showed evidence of peak activity within the cells up to5 hrs indicating the presence of ¹⁴C-compound 17. The other time periodsgave no peaks as the signal to noise ratio was too low which indicatesthat ¹⁴C-compound 17 has been broken down within the cell. 0.1% SDS wasused as a test to see if the extraction process from the cells causedthe degradation of ¹⁴C-compound 17. The analysis of ¹⁴C-compound 17 inMEM and SDS however showed that it is quite stable within thesesolutions up to 5 hrs.

The conclusion can be reached that ¹⁴C-compound 17 is stable enoughwithin these environmental conditions to have been taken up within thecells and is consequently broken down.

Autoradiography experiments demonstrate that both ¹⁴C-ascorbic acid and¹⁴C-compound 17 are associated with the mineralising nodules and not thesurrounding confluent osteoblast monolayer.

EXAMPLE 9 In Vivo Biodistribution of ¹⁴C-compound 17

Method

Three rats were anaesthetised with halothane and injected with 0.2 ml¹⁴C-compound 17 in PBS (4 μCi total dose; specific activity 5 mCi/mmol).After 60 minutes, the rats were sacrificed by cervical dislocation.Tissues were placed into pre-weighed glass scintillation vials and 3 mltoluene added. Samples were incubated overnight at 50° C. Samples werethen decolourised with 0.1 ml Na-EDTA:0.5 ml H₂O₂ overnight at 50° C.Finally 10 ml Hionic-Fluor (Packard) was added to each vial and samplescounted on a LKB scintillation counter.

Results

In this preliminary study, most of the activity was present in theblood, liver and kidneys after 60 minutes. Measurement of activitypresent in the femur showed 0.041% id/g in the diaphysis and 0.095% id/gin the epiphysis, a ratio of 2.3 demonstrating greater uptake into theactively growing tips of the bone.

HPLC analysis showed the compound to be stable in plasma, hence it islikely that the uptake seen is due to intact ¹⁴C-compound 17.

What is claimed is:
 1. A compound comprising:

wherein: X¹ is OH or SH or NH₂ or —L-Z; X² and X³ are the same ordifferent and each is H, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, benzyl, aprotecting group or —L-Z; X⁴ is H or C₁₋₄ alkyl; L is a linkercomprising a chain of 0-10 atoms; Z is a group comprising a detectablemoiety that excludes ³H, ¹⁴C, ¹⁸F, and ¹¹C; and wherein when neither ofX2 or X3 is —L-Z that the compound further includes at least onedetectable moiety.
 2. The compound of claim 1, wherein the detectablemoiety comprises a metal complex of a chelating agent.
 3. The compoundof claim 1, wherein the detectable moiety is a radionuclide.
 4. Thecompound of claim 3, wherein the radionuclide is a gamma emitter.
 5. Thecompound of claim 4, wherein the gamma emitter is ^(99m)Tc or ¹²³I. 6.The compound of claim 3, where the radionuclide is ¹²³I, ¹²⁵I or ¹³¹Iand Z is an iodovinyl group, or an iodo-C₅₋₁₂-aryl group.
 7. Thecompound of claim 1, where the detectable moiety is a hyperpolarisedatom.
 8. The compound of claims 1, wherein X¹ is —L-Z.
 9. The compoundof claims 1, wherein L is a linker group which comprises a 0-10 atomchain and has the formula (A)_(m) where A is —CR₂—, —CR═CR—, —C≡C—,—NRCO—, —CONR—, —O(CO)—, —(CO)O—, —SO₂NR—, —NRSO₂—, —OCR₂—, —SCR₂—,—NRCR₂—, a C₄₋₈ cycloheteroalkylene group, a C₄₋₈ cycloalkylene group, aC₅₋₁₂ arylene group or a C₃₋₁₂ heteroarylene group; m is an integer ofvalue 0 to 10; each R group is independently chosen from H, C₁₋₄ alkyl,C₁₋₄ alkenyl, C₁₋₄ alkynyl, C₁₋₄ alkoxyalkyl or C₁₋₄ hydroxyalkyl. 10.The compound of claim 9, wherein the linker group comprises a 0-4 atomchain.
 11. The compound of claims 1, of formula:

with X¹-X⁴, L and Z as defined in claim
 1. 12. The compound of claim 11,wherein each of X² and X³ is H.
 13. A method of diagnosing metastaticbone disease using compound of claim 1, comprising: providing a unitdose of the compound; injecting a unit dose into a patient; and imagingsaid patient to identify uptake of compound by activated osteoblasts atsites of increased bone turnover.
 14. A method of using the compound ofclaim 1 in the radiotherapy of metastatic bone disease, comprising:providing a unit dose of the compound; injecting a unit dose into apatient; and delivering a local targeted radioactive dose to activatedosteoblasts at sites of increased bone turnover.